GC–MS and LC-TOF–MS profiles, toxicity, and macrophage-dependent in vitro anti-osteoporosis activity of Prunus africana (Hook f.) Kalkman Bark

Osteoporosis affects millions of people worldwide. As such, this study assessed the macrophage-dependent in vitro anti-osteoporosis, phytochemical profile and hepatotoxicity effects in zebrafish larvae of the stem bark extracts of P. africana. Mouse bone marrow macrophages (BMM) cells were plated in 96-well plates and treated with P. africana methanolic bark extracts at concentrations of 0, 6.25, 12.5, 25, and 50 µg/ml for 24 h. The osteoclast tartrate-resistant acid phosphatase (TRAP) activity and cell viability were measured. Lipopolysaccharides (LPS) induced Nitrite (NO) and interleukin-6 (IL-6) production inhibitory effects of P. africana bark extracts (Methanolic, 150 µg/ml) and β-sitosterol (100 µM) were conducted using RAW 264.7 cells. Additionally, inhibition of IL-1β secretion and TRAP activity were determined for chlorogenic acid, catechin, naringenin and β-sitosterol. For toxicity study, zebrafish larvae were exposed to different concentrations of 25, 50, 100, and 200 µg/ml P. africana methanolic, ethanolic and water bark extracts. Dimethyl sulfoxide (0.05%) was used as a negative control and tamoxifen (5 µM) and dexamethasone (40 µM or 80 µM) were positive controls. The methanolic P. africana extracts significantly inhibited (p < 0.001) TRAP activity at all concentrations and at 12.5 and 25 µg/ml, the extract exhibited significant (p < 0.05) BMM cell viability. NO production was significantly inhibited (all p < 0.0001) by the sample. IL-6 secretion was significantly inhibited by P. africana methanolic extract (p < 0.0001) and β-sitosterol (p < 0.0001) and further, chlorogenic acid and naringenin remarkably inhibited IL-1β production. The P. africana methanolic extract significantly inhibited RANKL-induced TRAP activity. The phytochemical study of P. africana stem bark revealed a number of chemical compounds with anti-osteoporosis activity. There was no observed hepatocyte apoptosis in the liver of zebrafish larvae. In conclusion, the stem bark of P. africana is non-toxic to the liver and its inhibition of TRAP activity makes it an important source for future anti-osteoporosis drug development.

www.nature.com/scientificreports/ Osteoporosis is a silent but one of the major global health problems characterized by deterioration of bone microarchitecture and low bone mass 1,2 . It is one of the leading causes of morbidity in older people above 40 years 3 . This condition occurs when the rate of bone resorption is higher than the rate of bone formation and consequently presents a greater risk of fractures for the persons suffering from it. Factors such as aging, sex steroid deficiency, and as well as menopause in women have been associated with a higher risk of osteoporosis 4 . Activated macrophages have also been implicated in the pathogenesis of osteoporosis by stimulating the development of osteoclastogenesis-associated bone loss 5 . Cytokines such as tumor necrosis factor alpha (TNFα), interleukin-1 (IL-1β) and interleukin-6 (IL-6) are pro-inflammatory and play central role in inflammation of which IL-6 is the most important in chronic inflammatory and autoimmune diseases, cytokine storm and cancer 6,7 . IL-6 is fundamental in a number of processes including bone metabolism, inflammation, hematopoiesis 7 . IL-6 is also implicated in mediation of IL-1 effects, a potent bone resorption stimulator 8 . Although a number of conventional methods have been employed in the treatment of osteoporosis including bisphophates and estrogen hormonal therapy, adverse effects associated with these therapies that are reported to limit their use include; gastrointestinal tract disturbances and burning sensation 9 . Exploration of other avenues including the use of natural products in the treatment of osteoporosis have been suggested to offer a better alternative with lesser adverse side effects 9 . Herbal medicines have been used over the years to prevent and treat osteoporosis condition 2 . The anti-osteoporosis of herbal products has been attributed to the secondary metabolites including alkaloids, terpenes, steroids, and phenolic compounds 10,11 and these have led to the development of a number of drugs over the years with great therapeutic activities 12 . Prunus africana (Hook f.) Kalkman (Family Rosaceae), commonly called African cherry is an evergreen plant endemic in sub-Saharan Africa 13 , and contains a number of secondary metabolites like terpenes, alkaloids, phenolic compounds, and sterols in its stem bark 14 . For centuries, P. africana has been used in Africa to treat myriad of diseases including prostate cancer, hyperplasia, diabetes, malaria, and inflammatory conditions 15 . Despite its immense medicinal uses and with a wide array of secondary metabolites, P. africana has not been investigated for anti-osteoporosis activity yet it is used by the persons with prostate cancer who are vulnerable to suffer from osteoporosis 16,17 . Hence, this study evaluated the phytochemistry P. africana bark extract and it's in vitro anti-osteoporosis activity based on osteoclast tartrate-resistant acid phosphatase (TRAP) as a cytochemical marker of osteoclasts. However, due to the toxicity associated with some of the herbal medicines such as liver damage 18 and owing to association of liver health and osteoporosis 19 , this study also evaluated the hepatotoxicity of P. africana bark extracts in zebrafish (Danio rerio) larvae.

Materials and methods
Plant material and preparation of extract. The study was conducted in accordance to the relevant institutional, national, and international guidelines and legislation. The stem bark of P. africana (1 kg) was obtained from P. africana tree (Fig. 1a,b) in the herbal garden of Natural Chemotherapeutics Research Institute, Ministry of Health, Uganda. The study material was identified by Dr. Sungyu Yang at Korea Institute of Oriental medicine (KIOM) and a voucher specimen (number KIOM201901022377) of the sample was deposited in the Korean Herbarium of Standard Herbal Resources (Index Herbarium code: KIOM) at KIOM, South Korea. The stem bark (Fig. 1c) was dried in an oven at 40 °C and then ground using a steel pulverizing machine (250G New Type Pulverizing Machine, Model RT-N04-2 V, Taiwan) to obtain a fine powder (Fig. 1d). 500 g of the fine powder sample was extracted by maceration using 1,500 ml of methanol. The extract was filtered using Whatman filter No. 1 after 24 h. and concentrated under a vacuum reduced pressure at 40 °C, 70 rpm, using an EYELA N-1200B (Tokyo Rikakikai Co. Ltd, Japan) efficient rotary evaporator. The concentrated extract was then vacuum dried and yielded 60 g of extract. The resultant dried extract was then used in the subsequent TRAP assay, cell viability assay, and experiments on the production of inflammatory factors.
Gas chromatography-mass spectrometry (GC-MS) sample preparation and analysis. The P.
africana bark sample was extracted in 100% methanol by sonication for 30 min. The extract was then prepared at 50 µg/L; filtered through a 0.2 μm syringe membrane filter from Whatman Ltd (Maidstone, UK) and subjected to GC-MS analysis. The analysis was performed in a 7890B GC-MS system (Agilent Technologies, Atlanta, GA, USA), coupled with a 7977B model mass detector (Agilent Technologies, Atlanta, GA, USA) using DB-5 MS capillary column (30 m × 0.25 mm × 0.25 μm). Chromatographic conditions were as follows: the extract (1 μL) was injected in split mode with a ratio of 1/20 at 250 °C; oven initial temperature was 50 °C and increased 110 °C during 5 min, followed by heating at a rate of 7 °C/min at 300 °C. The mass analyzer was set to scan from 30 to 600 amu. Peak identification was carried out by comparison of the experimental mass spectrum in the National Institute of Standards and Technology (NIST) and Wiley GC-MS libraries.
Liquid chromatography time-of flight mass spectrometry (LC-TOF-MS) analysis. The P. africana bark sample was extracted and prepared as in "Gas chromatography-mass spectrometry (GC-MS) sample preparation and analysis" above. The LC-TOF-MS analysis was performed on an Agilent 1290 infinity II system coupled with an AB SCIEX Triple TOF 5600 mass spectrometer equipped with electrospray ionization. Gemini® C 18 (250 mm × 4.6 mm i.d., 5 μm, Phenomenex, USA) was used for column separation. The column temperature was maintained at 40 °C, the flow rate was 1.0 ml/min, and the injection volume was 10 μl. The optimal mobile phase consisted of a linear gradient system of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile, 0-2 min, 3% B; 2-30 min, 3 TRAP assay and BMM cell viability. This was performed following the previously described method 21 . The measurement of osteoclast TRAP activity was based on the generation of absorbance by incubating BMM cells with TRAP buffer (50 mM sodium tartrate, 0.12 M sodium acetate, pH 5.2) and p-nitrophenyl phosphate (1 mg/ ml) for 15 min. For TRAP staining, the BMM cells were incubated with TRAP buffer containing naphthol AS-MX phosphate (0.1 mg/ml) and Fast Red Violet (0.5 mg/ml). The BMM cells were then cultured with P. africana methanolic extract at 0, 6.25, 12.5, 25, and 50 µg/ml concentrations and 50 μM of β-sitosterol, chlorogenic acid, catechin and naringenin each in the presence of RANKL for 6 days. The osteoclast TRAP activity was determined using a colorimetric assay with p-nitrophenyl phosphate as a substrate. The cell viability was deter- www.nature.com/scientificreports/ mined using Cell Counting Kit-8 (CCK) (WST-8/CCK8; Dojindo), according to the manufacturer's instructions.
For the measurement of cell viability, cells were plated in 96-well plates and treated with P. africana extracts concentrations as above for 24 h. After incubating with the CCK solutions and the cells for 1 h, the absorbance was measured at 450 nm using a microplate reader (Versa Max) and results were presented as a percentage of the vehicle control.

Nitrite (NO) assay and RAW 264.7 cell viability. Murine macrophage RAW 264.7 cells were cultured
in DMEM medium supplemented with 100 U/mL of penicillin, 100 ug/mL of streptomycin, and 10% heat-inactivated FBS. The nitrite concentration in the supernatant from cultured cells was analyzed using the Griess reaction test. RAW 264.7 cells were plated at a density of 5 × 10 4 cells/mL in 96-well culture plates, pre-incubated with samples (P. africana methanolic extracts (150 μg/mL), β-sitosterol, chlorogenic acid, catechin and naringenin (100 μM each) for 3 h, and stimulated with LPS (200 ng/ml) for 24 h. Griess reagent (1% sulfanilamide, 0.1% N-1-napthylethylenediamine dihydrochloride, and 2.5% phosphoric acid) was mixed with an equal volume of cell supernatant, and absorbance was measured at 570 nm using the ELISA reader. Sodium nitrite was used as a standard. Dexamethasone was used as a positive control (40 or 80 μM).

Enzyme-linked immunosorbent assay (ELISA). The concentrations of the inflammatory cytokines
IL-1β (R&D, USA) and IL-6 (MyBiosource, USA) in culture supernatant was determined using ELISA antibody kits following the manufacturer1s protocol (MyBiosource, USA). RAW264.7 cells were grown in 96-well culture plates at a density of 5 × 10 4 cells/mL, pre-incubated with samples for 3 h, and stimulated with LPS for 6 h (IL-1β) or 24 h (IL-6). The cytokines produced in each sample were calculated from standard curves using known concentrations of recombinant cytokines for each ELISA antibody kit.
Hepatotoxicity assay in zebrafish (Danio rerio) larvae. This was performed following the previously described method 21 . All methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by Korea Research Institute of Chemical Technology research ethics committee and conducted in compliance with the ARRIVE guidelines. Zebrafish larvae were used for this study as previously described 22 . At 96 h post-fertilization (hpf), the larvae were transferred to a transparent 24-well plate (N = 10/well) with 1 ml of embryonic medium. The larvae were then exposed to increasing concentrations of 25, 50, 100, and 200 µg/ml of P. africana methanolic and ethanolic extracts and water extract from 90 to 120 hpf. Dimethyl sulfoxide (DMSO) was used as a negative control while 5 μM of tamoxifen (Sigma-Aldrich, St. Louis, MO, USA) was used as a positive control. To obtain images, the larvae were anesthetized in tricaine (Sigma-Aldrich), mounted in 3% methyl cellulose (Sigma-Aldrich), and observed under a Leica MZ10 F stereomicroscope equipped with a Leica DFC425 camera and Leica application Suite software (version 4.5).
Statistical analysis. Data were represented as the mean ± standard deviation. Statistical significance between groups was analyzed using the Student's t-test and p-values < 0.05 were considered statistically significant.

Results
GC/MS analysis. The GC/MS analysis of the P. africana extract was based on mass spectra, retention times, and quality ratio analysis revealed the presence of 32 components (Table 1)

LC-TOF-MS analysis.
Based on the chemical profiling by LC-TOF-MS analysis, the various phytochemicals were detected from the P. africana extract. In the positive ion mode, as a result of analysis using retention index libraries, 65 peaks with a library score over 90% were identified from the P. africana extract (Supplementary Table Y). Among them, 24 components showed reliable mass (over 98% of library score) in the P. africana extract, especially, 7 components (Astragalin, Chlorogenic acid, Coproporphyrin I, Hyperin, Luteoloside, Mesoporphyrin IX and Naringenin), showed the accurate mass according to result of 100% library score ( Table 2). In the negative ion mode, 72 peaks with a library score over 90% were identified from the P. africana extract (Supplementary Table Z). Among them, 29 components showed the reliable mass (over 98% of library score) in the P. africana extract, especially, 5 components (Pedunculoside, Luteoloside, Hexadecanedioic acid, Guanosin, Betulonic acid and Naringenin), showed the accurate mass according to result of 100% library score ( Table 2). In the present study, among the identified primary and secondary metabolic components, Catechin showed the largest peak area among the identified primary and secondary metabolic components in the P. africana extract (Supplementary Table Y Fig. 2A).
Prunus africana methanolic stem bark extract had a significant (p < 0.05) simulative effect at concentrations of 12.5 and 25 µg/ml on the cell viability of BMM cells (Fig. 2B) compared to the control. However, at a higher concentration of 50 µg/ml the methanolic stem bark extract, the cell viability reduced to 70% compared to the control. The high viability of the BMM cells may indicate the non-cytotoxicity of P. africana bark.
The effects of β-sitosterol, chlorogenic acid, catechin and naringenin (50 μM each) on RANKL-induced TRAP activity representing osteoclastogenesis were evaluated. We found that these compounds significantly inhibited RANKL-induced TRAP activity without showing cell toxicity (Fig. 3).
Inhibitory effect on NO production. Since NO production is correlated with various inflammatory diseases, we determined the suppressive effects of samples (P. africana (PA) methanolic extract, β-sitosterol, chlorogenic acid, catechin and naringenin) on NO levels in RAW264.7 cells via LPS stimulation. To determine NO levels in the supernatant, cells were pre-treated with samples for 3 h, followed by stimulation with LPS for 24 h, and then measured using Griess reagent. As the positive control, dexamethasone showed strong suppressive effect on NO secretion upon LPS stimulation. All the investigated samples dramatically inhibited NO production after LPS stimulation (Fig. 3b). All samples did not significantly affect cell viability and β-sitosterol that increased it (Fig. 3a).
Inhibitory effect on IL-6 and IL-1β levels. The effects of samples on inflammatory cytokine, IL-6, secretion in macrophages were evaluated using enzyme-linked immunosorbent assay (ELISA). IL-6 secretion was significantly inhibited by PA-methanol (p < 0.001) and β-sitosterol (p < 0.0001) (Fig. 3c). Chlorogenic acid and naringenin, but not catechin, significantly inhibited LPS-induced IL-1β level as shown in Fig. 3d. Hepatotoxicity in zebrafish larvae. In this study, the zebrafish larvae exposure was done from 96 to 120 hpf and those exposed to DMSO showed no liver cell death (shown by white dash line) (Fig. 4A) but tamoxifen treatment resulted in liver cell death (shown by red dash line) (Fig. 4B). At a concentration of 50 and 100 µg/ ml water extract of P. africana, 30% and 10% of the zebrafish larvae survived at 120 hpf respectively and death of hepatocytes was not observed in them (Fig. 4C). However, at a higher concentration of 200 µg/ml P. africana water extract, 100% larvae mortality was observed before 120 hpf. At a concentration of 25 µg/ml ethanolic extract, 50% of the zebrafish larvae survived and hepatocytes death was not observed in them at 120 hpf (Fig. 4D). However, at higher concentrations of 50 and 100 µg/ml, 100% larvae mortality rate was observed at 120 hpf. 100% larvae mortality was observed for larvae exposed to P. africana bark methanolic extracts at various concentrations of 25, 50, and 100 µg/ml before the 120 hpf.

Discussion
TRAP is a specific and reliable cytochemical marker used as a measure of activated macrophages 23 . The bone marrow macrophages play a major role in the activation and formation of osteoclasts and hence important for the pathogenesis of osteoporosis 5 . Osteoporosis reflects increased osteoclast function relative to that of osteoblasts and hence the pharmacological arrest of osteoclasts is a mainstay in the treatment of systemic bone loss 24 . TRAP activity is an important cytochemical marker of osteoclasts and its concentration in the serum is utilized as a biochemical and histochemical marker of osteoclast function and degree of bone resorption 25 . Therefore, as a measure of osteoclast number and bone resorption, TRAP plays a vital role in osteoporosis diagnosis and prognosis 23 . And as evidenced in the previous study, the suppressing of TRAP pathway prevented ovariectomyinduced osteoporosis in vivo 26 . Therefore, the significant (p < 0.001) inhibition compared to the control at concentrations of 6.25, 12.5, 25, and 50 µg/ml methanolic P. africana stem bark extracts is an indication of P. africana anti-osteoporosis effects. Additionally, chlorogenic acid, catechin, naringenin and β-sitosterol that are present www.nature.com/scientificreports/ in P. africana stem bark have been demonstrated in our study to remarkably inhibit RANKL-induced TRAP activity explaining indeed the antiosteoporotic potential of the plant stem bark. The anti-osteoporosis activity of P. africana bark extracts may be attributed to some of the compounds present in it including astragalin, hyperin, luteoloside, mesoporphyrin, naringenin, chlorogenic acid, β-sitosterol and catechin (Tables 1 and 2, and supplementary Tables Yand Z). Additionally, previous studies have also showed that P. africana stem bark is indeed rich in these compounds with anti-osteoporosis activity (Table 3). Astragalin demonstrated estrogenic anti-osteoporosis activity and significantly increased proliferation in osteoblastic cells (UMR-106) 27 . In another study, Astragalin promoted differentiation in MC3T3-E1 osteoblastic cells through activation of MAPK and BMP pathways and promoted in vivo bone formation 28 . Further, a number of other studies have showed anti-osteoporosis activity of Astragalin 10,29,30 , and its presence in P. africana stem bark as confirmed in our study shows that it indeed contributes to the anti-osteoporosis activity of the plant stem bark. Hyperin was one of the major 3 chemical compounds in Cuscuta chinensis that showed anti-osteoporosis effects 29 . In fact in their study, Tao, et al. 29 ascribed the anti-osteoporosis effect of C. chinensis to hyperin as a result of its high positive correlation to anti-osteoporosis activity. Hyperin was also reported to markedly increase alkaline phosphatase (ALP) activity in osteoblast cells 31 .
Luteoloside, a natural compound is known to suppress activity of osteoclasts thus could potentially be used for treating bone metabolism disorders including osteoporosis 32 . Luteoloside in a previous study was showed to possess strong inhibition against LPS induced osteolysis in an in vivo study 32 . Further, it was also demonstrated to suppress differentiation of RANKL-induced osteoclast and decrease bone resorption tendency dose dependently 32 . The anti-osteoclastic and anti-resorptive actions of luteoloside were not only through blockage of NFATc1 activity and debilitation of RANKL-mediated Ca 2+ signaling but also through MAPK and NF-κB pathways 32 . Mesoporphyrin, a porphyrin derivative has been reported to possess anti-inflammatory activity through inhibiting IL-6 production 33 . Since IL-6 potently activates osteoclasts and is responsible for bone resorption 34 , mesoporphyrin IX may thus possess anti-osteoporosis activity through inhibition of IL-6 production. www.nature.com/scientificreports/ Naringenin has been reported to significantly inhibit osteoclastogenesis with inhibitions of up to 96 ± 1% at 50 μg/ml 35 . Naringenin has also been demonstrated to not only significantly inhibit secretion of monocyte chemoattractant protein-1, interleukin (IL)-1α and IL-23 but also markedly decrease release of a bone resorption activity indicator, helical peptide 620-633, thus greatly inhibiting osteoclastic bone resorption and human osteoclastogenesis 35 . La, et al. 35 indicates that naringenin could be used to treat bone-related diseases such as Table 3. Summary of the previous studies on anti-osteoporosis effects of some of the compounds identified in P. africana.

S/No
Phytochemical compounds Compound structure Anti-osteoporosis effects Reference www.nature.com/scientificreports/ osteoporosis. In the current study, naringenin was observed to significantly inhibit LPS-induced NO production to a level even greater than the effect of dexamethasone (positive control) and also remarkably suppress IL-1β generation. Since increased NO level inhibits growth and differentiation of osteoblasts 36 , and IL-1β stimulates bone resorption 37 , naringenin suppression of NO and IL-1β production signifies that it is important in preventing osteoporosis. In another study, naringenin was showed to significantly promote osteogenic differentiation 38 . Naringenin being present in the stem bark of P. africana contributes to its anti-osteoporosis activity. Catechin was identified as possessing the strongest osteogenic effects from a batch of herbal ingredients used in traditional Chinese medicine using human mesenchymal stem cells (hMSCs) 39 . Catechin was reported to increase the activity of ALP, deposition of calcium and Runx2 mRNA expression among others 39 . It was thus proposed to enhance osteogenesis through increasing protein phosphatases 2A (PP2A) level inhibiting extracellular signal-regulated kinase (ERK) signaling in hMSCs 39 . In another study, catechin rich extract was demonstrated to promote formation and enhance survival of osteoblasts and inhibiting the activity and growth of osteoclasts 40 . Similar to our findings, catechin in previous studies has been documented to suppress NO production in LPS stimulated macrophages 41,42 . Thus, our study suggests that catechin exerts antiosteroporotic effect by eliminating the inhibitory effect of NO on growth and differentitation of osteoblasts. The presence of catechin in the stem bark of P. africana enhances its anti-osteoporosis potential. Vanillic acid has been reported to exhibit anti-osteoporotic activity by inhibitory effects on bone resorption 48 ; improving bone mineral density and bone mineral content and as well as biomechanical stability 43 and protecting trabecular structure from degradation by osteoclasts in ovariectomized postmenopausal mice 44 . Sorbitol; a sugar alcohol with a sweet taste has been observed to retard bone resorption in Sprague Dawley male rats 45 . The positive effects on osteoporosis prevention by fruits including that of Prunus domestica and Prunus salicina may partly be due to the presence of sorbitol compound in them 1 . Recent studies suggest that inflammation is one of the key factors that influence bone turnover, leading to osteoporosis 3 . Therefore, the potent anti-inflammatory activity of β-sitosterol 47 ; a compound present in P. africana stem bark may further explain its anti-osteoporosis activity. Stearic acid and oleic acid have been reported to inhibit osteoclastogenesis in bone marrow cultures and RAW264.7 cells 46 . Chlorogenic acid was observed to improve the bone quality by modifying the bone mineral density and trabecular microarchitecture in an ovariectomy rat model 49 . Furthermore, chlorogenic acid was also observed to promote proliferation of osteoblast precursors and osteoblastic differentiation in ovariectomized rats 50 . In the present study, chlorogenic acid exhibited significant inhibition of LPS-induced NO production and secretion of IL-1β. Interleukin-1 is a very powerful stimulator of bone resorption and is well known to inhibit bone formation 51 . The significant inhibition of IL-1β and NO production by chlorogenic acid indicates that it plays an important role in preventing and treating osteoporosis. In addition, our study showed that chlorogenic acid had a significant inhibition of RANKL-induced TRAP activity giving further evidence of its antiosteoporotic potential.
In light of these results, the inhibition of the TRAP activity by P. africana bark extracts may therefore be due to these different important compounds in it including chlorogenic acid, catechin, naringenin and β-sitosterol. These findings therefore provide valuable insight in to the anti-osteoporosis potential of P. africana.
Excess production of NO in the body system plays a vital part in pathogenesis of inflammatory diseases including osteoporosis 21 . It has been reported previously that increased production of NO is a contributing factor to osteoporosis pathogenesis 21,52 . Thus, a potential therapeutic pathway for managing the disease is through suppression of NO production as indicated by Komakech, et al. 21 . In this study, the P. africana methanolic extract (each 150 µg/ml) significantly (p < 0.0001) inhibited NO production actually more than the positive standard, dexamethasone (40 µM). The chemical compounds, chlorogenic acid, catechin, naringenin and β-sitosterol (100 µM each) in RAW264.7 cells also significantly suppressed LPS-induced NO production. Studies have reported that high NO concentrations have great inhibitory effects on growth and differentiation of osteoblasts 36,[53][54][55] . This has been suggested to be partly as a result of NO pro-apoptotic effects on osteoblasts 56 . The significant inhibition of LPS-induced NO production by PA extracts and its chemical constituents clearly demonstrates its antiosteoporotic potential by eliminating the inhibitory effects of NO to growth and differentiation of osteoblasts. Chlorogenic acid, catehin, naringenin and β-sitosterol did not show toxic effects.
In our current study, PA-methanolic extract (150 µg/ml) and β-sitosterol (100 µM) just like the positive control (dexamethasone [40 µM and 80 µM]) in RAW264.7 cells were observed to significantly inhibit LPS-induced IL-6 secretion. This therefore suggests that methanol extracted phytochemical compounds from P. africana stem bark including β-sitosterol exhibited antiosteoporotic potential by inhibiting the production of IL-6 within the bone microenvironment. IL-6 is known to potently activate osteoclasts and is responsible for bone resorption 34 . Pro-inflammatory cytokines notably IL-6 are vital in normal processes of bone remodeling and pathogenesis of osteoporosis in elderly persons and during perimenopause 34 . Production of IL-6 induces eminent lytic lesions along with diffuse osteoporosis typical of the disease 34 . Before menopause, estrogen in bone marrow regulates the expression of most notably IL-6 34 . IL-6 levels are known to increase with age in not only humans but monkeys and mice and in the trend with osteoporosis 34 .
Liver toxicity from herbal and dietary supplements is a common phenomenon and is a leading cause of a number of underlying liver diseases 57 . Osteoporosis is a frequent complication in patients with liver complications 58 . Indeed, decreased trophic factors such as insulin growth factor in the liver due to liver toxicity or chronic diseases including diabetes my result in osteoblast dysfunction 19 . Therefore, ensuring a healthy liver is fundamental in maintaining a balanced body biological processes including prevention of bone loss. Zebrafish larvae is an important model system for the evaluation of the liver toxicity when an organism is exposed to a toxicant 59 . Liver organogenesis in zebrafish initiates at 30 hpf on the left-hand side of the embryo and an enlarged liver bud connects with the intestine and functionally matures until 72 hpf 60 . Treatment of the zebrafish larvae with liver toxicants such as tamoxifen reduces liver transparency due to the liver cell death 61  www.nature.com/scientificreports/ 100 µg/ml water extract and 25 µg/ml ethanolic extract was used to determine hepatotoxicity in zebrafish larvae. And although each experimental group showed larval mortality, the P. africana extracts did not induce hepatotoxicity because living larvae did not show liver-specific cell death. Considering that the extraction method can influence the composition of extracts 62 , this may explain difference in the mortality rate of the zebrafish larvae in the methanolic and water extracts of the P. africana stem bark. Previous studies also showed that P. africana bark extract administered at 1000 mg/kg body weight had no visible deleterious effects on BALB/c mice 63 and showed mild hepatotoxicity and nephrotoxicity in Sprague-Dawley rats 64 . These observations therefore showed that the stem bark of P. africana its non-toxic within a low dose range.

Conclusions
The macrophage-dependent anti-osteoporosis activity of P. africana bark may be attributed to the synergistic action of the various phytochemicals in its stem bark including chlorogenic acid, catehin, naringenin, vanillic acid, sorbitol, octadecanoic acid (stearic acid), and β-sitosterol. NO production was significantly inhibited (all p < 0.0001) by P. africana methanolic, chlorogenic acid, catehin, naringenin, and β-sitosterol. IL-6 secretion was significantly inhibited by P. africana methanolic extract (p < 0.0001) and β-sitosterol (p < 0.0001) and in addition, chlorogenic acid and naringenin remarkably inhibited IL-1β production. All samples displayed significant inhibition of RANKL-induced TRAP activity. Although the methanolic extract of P. africana bark exhibited potent anti-osteoporosis activity, we recommend that future studies should carry out isolation of the individual chemicals or group of chemicals that are/is responsible for its anti-osteoporosis activity. Nonetheless, this study has demonstrated that P. africana bark extracts have no overt hepatotoxic effects in zebrafish larvae at a given dose range and offers a basis for future studies and medicine development with anti-osteoporosis therapeutic application.

Data availability
The data for this current study are available from the corresponding author upon reasonable request.